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GeneArt® It! 3
Gene optimization 10
Expression-ready genes 13
Whether you need industry-leading gene synthesis services or optimized protein expression, or want to outsource the entire process from gene synthesis
to protein and cell line production, this brochure outlines the GeneArt® products and services that can help you succeed.
Utilize sequencing platforms Apply rational design Whether you outsource to us From small-scale research to
to generate knowledge and software to optimize gene or do it yourself, select from industrial production in applied
analyze cellular data (using expression (using GeneArt® a leading line of GeneArt® markets, put it all together with
Vector NTI® sequence analysis GeneOptimizer ® software) products (such as gene a complete toolbox of products
and design software, and Ion synthesis tools) and services and services for superior cell
Torrent™ or SOLiD® Systems) (such as subcloning and line development and growth,
plasmid preparation) for optimal as well as protein expression
speed, quality, and performance and production
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GeneArt It! ®
We are your partner for gene synthesis through protein and cell production
GeneArt® Gene Synthesis GeneArt® Strings™ GeneArt® custom services GeneArt® Directed Evolution
A reliable and cost-effective DNA Fragments Whether you’re looking to save Directed evolution can be used
method for obtaining customized A time-saving alternative to PCR, time or improve upon existing to create biomolecules for basic
DNA constructs with 100% GeneArt® Strings™ DNA Fragments processes, GeneArt® services research, medical science, and
sequence accuracy, GeneArt® Gene are available up to 3 kb and are provide: industrial production. Results
Synthesis offers: compatible with any downstream • A single resource for all of can be achieved quickly, with just
• The largest capacity cloning method of choice, providing: your outsourcing needs a few rounds of mutagenesis and
and fastest production • An economical solution that selection. GeneArt® Directed
• Gene synthesis, custom cell lines, Evolution services include:
processes maintains the gene synthesis
and mammalian/baculovirus
benefits of both flexibility and • GeneArt® combinatorial libraries
• Outstanding quality— protein production
performance
ISO 9001:2008 certification • GeneArt® site-directed
• Cloning and plasmid prep
• Easy ordering—you can directly mutagenesis
• Gene optimization for services
enter, edit, optimize, and order
reliable maximum protein
your sequence through the online • ISO 9001:2008 and ISO
expression
GeneArt® portal 13485:2003 certification and
responsive project management
Facilities overview
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GeneArt Gene Synthesis ®
*Production time is the number of business days required to synthesize GeneArt® genes in our manufacturing facility.
Delivery time is in addition to production time and depends on the destination of the shipment.
**There is an upgrade fee for Express and SuperSPEED deliveries.
Expression
GeneOptimizer®
optimization algorithm
GeneAssembler™
gene synthesis platform
Wild type GeneArt® GeneOptimizer®
DNA sequence improved sequence
Classic cloning
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Figure 1. GeneArt® Gene Synthesis and optimization are typically faster than classic cloning methods and can provide better results.
GeneArt® Strings™ DNA Fragments are custom-made, uncloned, double-stranded linear DNA fragments up to 3,000 bp in length, assembled from
synthetic oligonucleotides using the same high-quality process developed for GeneArt® Gene Synthesis (Figure 2). Strings™ DNA Fragments are delivered
dried and ready for resuspension, cloning, and screening to enable identification of the correct clone. Strings™ DNA Fragments are a fast and smart
alternative for getting your synthetic gene and clone into your expression plasmid (see Figure 3 for an application example).
*Depending on the nature of the sequence, production time can vary. Delivery times are in addition to the specified production times and depend on location.
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Design
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Production of DNA 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘
Oligonucleotide
fragments using synthesis
GeneArt® technology
Gene assembly
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63 63
60%
Downstream 56
assembly 50 50 50
40%
20%
0
624 698 844 960 984 2,545 2,725 2,894 2,946 2,980
Figure 2. Producing a synthetic gene using GeneArt® Strings™ DNA Fragments. Figure 3. Ten Strings™ DNA Fragments up to 3,000 bp were cloned using the GeneArt®
Seamless Cloning and Assembly Kit. The figure shows the percentages of clones with
correct length as identified by colony PCR (cloning efficiency), and the percentages of
correct-sequence clones (fidelity) based on the number of full-length clones.
Experience enhanced protein expression based on gene optimization GeneOptimizer® sequence processing includes the following:
Production of recombinant human proteins in human cells for • Identification of the best way to incorporate your requested
biomedical research and product development can be hampered by low sequence elements
expression yields. Similarly, transient protein expression in any research
• Elimination of cryptic splice sites and mRNA-destabilizing
lab is at risk of being insufficient, especially (but not exclusively) for
sequence elements for increased mRNA stability
heterologous expression. These expression issues can limit researchers’
ability to conduct their structural and functional analyses, delaying (and in • Codon optimization and GC content adaptation for your
some cases halting) the discovery process or the entire research project. expression system
• Avoidance of undesirable mRNA secondary structures
Gene optimization is the solution to traditional protein expression
limitations. Common pain points associated with protein expression, such • Elimination of repetitive sequences
as yield, can now be addressed in a rational and systematic way. Using
• siRNA-resistant forms of wild type genes that can be used
data available from published literature in combination with proprietary
in RNAi rescue experiments
data, the GeneOptimizer® algorithm determines the optimal gene
sequence for your expression experiments (Figure 4). Optimization has
been experimentally proven to increase protein expression rates up to 100-
fold in a variety of host systems (Fath et al. 2011).†
†Stephan Fath, Asli Petra Bauer, Michael Liss, Anne Spriestersbach, Barbara Maertens, Peter Hahn, Christine Ludwig, Frank Schafer, Marcus Graf, and Ralf Wagner,
"Multiparameter RNA and Codon Optimization: A Standardized Tool to Assess and Enhance Autologous Mammalian Gene Expression,” PLOS One 6: 3, e17596.
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Up to a 100-fold increase in expression
The GeneOptimizer® process has been shown to help deliver large
increases in protein yield via a combination of factors that stabilize
mRNA and maximize translation efficacy (Figure 5). Efficacy has
been proven in a large comparative gene expression study (Fath et
al. 2011).†
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25 kDa
Protein 1
15 kDa
70 kDa
55 kDa
Standardized
Figure 4. The GeneOptimizer® software tool optimizes sequences for maximum transcription and protein load
translation efficiency.
Figure 5. Comparative expression analysis of wild type vs. an optimized human
gene. Western blot analysis using α-His antibody. Expression levels of 2 independent
transfections per wild type and optimized construct are displayed, showing a 3-fold
difference in expression in this particular analysis. Standardization is based on
endogenous protein.
must be <5 kb and must not contain complex sequences (optimization of GeneAssembler™ process
pMx pcDNATM 3.1*
your sequence with GeneOptimizer® software in the GeneArt® portal can
reduce complexity). Online Gene synthesis including cloning into pMX: Subcloning:
portal 9 business days 5–6 business days
Figure 6. Express cloning directly into an expression vector (top) compared with
classic subcloning from pMX into an expression vector (bottom).
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GeneArt Elements vector construction
® ™
Figure 7. The GeneArt® Elements™ vector construction template within the online GeneArt® portal.
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pCMV-IgK-leader+emGFP
pCMV-tPA-leader+emGFP
pCMV-GFP (cellular)
• Flexible—GeneArt® Elements™ DNA can be seamlessly assembled with
minimal design restrictions*
• Reliable—all offered parts are 100% sequence-verified
Mock
• Easy to use—intuitive CAD-like software within the GeneArt® portal
assists you with vector design
*Functionality in the final vector requires that you choose a resistance marker and an E. coli origin of replication from the GeneArt® Elements™ repository.
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To demonstrate the CPA process, we
generated a combinatorial library composed
of a small set of yeast promoters and
corresponding terminators from the GeneArt®
Elements™ part collection to analyze relative
luciferase expression levels (Figure 10). In
this experiment, the highest firefly luciferase
expression was shown using a TEF1
promoter/TEF1 terminator combination. This
demonstrates the proof of concept and can be
extrapolated to test other functional elements,
such as in metabolic pathways.
Figure 10. Analysis of a luciferase expression CPA experiment, comparing 25 combinations of the promoters and
terminators shown in Figure 9.
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F59A GVVPILVELDGDVNGHKFAVSGEGEGDATYGKLTLKFICT
F60A GVVPILVELDGDVNGHKFSASGEGEGDATYGKLTLKFICT
F61A GVVPILVELDGDVNGHKFSVAGEGEGDATYGKLTLKFICT
esis overnight
Combinatorial libraries
DNA sequences will be diversified using preassembled trinucleotides as building blocks 50 60 70 80
hipment (trinucleotide mutagenesis, or TRIM, technology) for the chemical synthesis process. wild type GVVPILVELDGDVNGHKXXVSGXGEXXATYGKLTLKFICT
peer A01 GVVPILVELDGDVNGHKRQVSGGGEGDATYGKLTLKFICT
This allows for the complete customization of the amino acid composition at randomized peer A02 GVVPILVELDGDVNGHKAGVSGEGEGDATYGKLTLKFICT
sites and thus avoids the occurrence of unwanted stop codons or amino acids. peer A03 GVVPILVELDGDVNGHKIYVSGLGEGDATYGKLTLKFICT
peer A04 GVVPILVELDGDVNGHKLKVSGPGEGDATYGKLTLKFICT
The deliverable is a pool of clones for your screening assay (e.g., phage display, mRNA
display). Receive the library as linear double-stranded PCR product (at least 2 μg of
DNA provided), or have the library cloned into the E. coli strain of your choice with
transformation rates of 109 or higher. With the cloned library, at least 30 μg of plasmid
50 60 70 80
DNA and 12 x 0.5 mL glycerol stocks will be provided.
wild type GVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLXXICT
peer A01 GVVPILVELDGDVNGHKFTVSGEGEGDATYGKLTLLKICT
peer A02 GVVPILVELDGDVNGHKTSVSGEGEDDATYGKLTLIGICT
peer A03 GVVPILVELDGDVNGHKFSVSGAGEGDATYGKLTLQDICT
Site-directed mutagenesis peer A04 GVVPILVELDGDVNGHKFSVSGEGEGYATDGKLTLLPICT
50 60 70 80
wild type GVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICT
F58A GVVPILVELDGDVNGHKASVSGEGEGDATYGKLTLKFICT
For other directed evolution services or benefits and applications, please go to lifetechnologies.com/directedevolution
F58C GVVPILVELDGDVNGHKCSVSGEGEGDATYGKLTLKFICT
F58D GVVPILVELDGDVNGHKDSVSGEGEGDATYGKLTLKFICT
F58E GVVPILVELDGDVNGHKESVSGEGEGDATYGKLTLKFICT
Online ordering
The GeneArt® Gene Synthesis sequence design and order portal
("GeneArt® portal") on our website offers intuitive and simple ordering
of your gene synthesis projects. This automated portal helps you
enter, edit, and optimize your sequences, and offers immediate price
quote information and ordering capabilities. Nearly all of the GeneArt®
services can be ordered using this intuitive ordering tool. You can even
perform in silico cloning and store your gene sequences, projects, and
personal vectors for future design. Save time and have full control and
flexibility with this one-stop shopping feature.
Figure 12. The Project Configurator within the online GeneArt® portal.
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Ordering GeneArt® Gene Synthesis: an example (Figure 13)
• Immediate ordering of wild type genes in the starting screen—just copy and
paste your sequence
• Choice of standard pMX vector or expression-ready vectors for Express cloning
• Quick procedure for sequence editing and full gene optimization
(optional, if wild type is not desired)
• Editing: include 5´ or 3´ cloning sites; add/delete/exchange sequences
• Optimization: choose expression organism, sequences to protect, and motifs
to avoid
• Downloadable sequence summary containing all customer requirements
• Add to cart and obtain price quote for easy ordering
Figure 13. An example order placed within the online GeneArt® portal. In this
example, Express cloning and plasmid preparation services have also been selected.
The GeneArt® portal can be used to order many GeneArt® products and
services, in addition to gene synthesis. These include GeneArt® Strings™ DNA
Fragments, GeneArt® Elements™, GeneArt® Precision TALs, and services
such as subcloning, plasmid preps, GeneArt® Directed Evolution, and custom
proteins and cell lines. Moreover, you can check the status of your web order(s)
in the manufacturing process—simply use the GeneObserver™ module within the
portal, available 24 hours per day.
Please see our library of videos on GeneArt® portal usage, including overview, setup, quick order,
optimization, and subcloning services, at lifetechnologies.com/genearttutorials
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Contact us for GeneArt® products and services
For further information or if you have questions, please don’t hesitate to contact us.
Email Europe America and Asia
geneartsupport@lifetech.com Phone: +49 (0) 941 942 76100 Phone: 800 955 6288 option 4/4/1